H3N2亚型流感病毒Vero细胞冷适应株遗传重配研究

发布时间：2018-07-11 13:47

本文选题：Vero细胞 + 冷适应　；参考：《北京协和医学院》2015年博士论文

【摘要】：流感病毒是严重威胁人类健康的一种急性呼吸道传染病病原体,每年在全球引起较高的发病率和死亡率。接种疫苗是预防和控制流感流行的最有效手段。目前批准使用的流感疫苗主要有三类：灭活苗、减毒活疫苗和重组HA疫苗。国内使用的仅有灭活的裂解疫苗或亚单位疫苗。流感减毒活疫苗以类似自然感染途径的喷鼻接种,既可刺激机体产生体液免疫,又能引起局部黏膜免疫和细胞免疫应答,其免疫效果安全有效。流感减毒活疫苗的制备主要是基于流感病毒的基因组为分节段的RNA,可以利用遗传重配技术将减毒供体株和流行株进行重配,重配株获得来自流行株的表面抗原基因和减毒株的表型特征。在该领域研究最多的是俄罗斯和美国,美国采用俄罗斯提供的冷适应供体株A/Ann Arbor/6/60(H2N2)和B/Ann Arbor/1/66制备的第一支三价流感减毒活疫苗Flumist(?)于2003年在美国上市。目前使用的流感减毒活疫苗均以鸡胚作为培养基质。以鸡胚作为基质生产的流感减毒活疫苗,存在外源因子污染和将鸡传染病病原体借由活疫苗接种播散到人群中的风险；同时鸡胚供应周期长,当大流感来袭时难以满足大规模生产需求,尤其是禽流感病毒来袭时鸡胚来源更受影响。鉴于此,WHO鼓励开展以哺乳动物细胞代替鸡胚作为流感疫苗生产基质的研究。Vero细胞是WHO批准生产人用疫苗的传代细胞系,但对流感病毒不敏感,产毒量不稳定。目前未见以Vero细胞为基质生产的季节性流感病毒减毒活疫苗,选育流感病毒Vero细胞冷适应株对于流感减毒活疫苗的研发至关重要。目前世界各国选育出的Vero细胞冷适应株极为稀少,仅见一株A/Singapore/1/57ca(H2N2)报道。本实验室经过梯度降温培养的方法选育出一株Vero细胞冷适应流感病毒株A/Yunnan/1/2005Vca(H3N2),已经在25℃连续传72代,病毒的滴度稳定在7.8-8.21gTCID5o/mL。经鉴定A/Yunnan/1/2005Vca(H3N2)具有冷适应表型(ca)、温度敏感表型(ts)和减毒表型(att),并且该毒株在雪貂和小鼠模型上表现出较好的安全性和免疫原性,能有效刺激机体产生细胞免疫和体液免疫,并能保护免疫动物抵抗同亚型流感病毒的致死性攻击,提示A/Yunnan/1/2005Vca(H3N2)有可能是制备流感病毒Vero细胞减毒活疫苗株的理想候选供体株。在本研究中,以实验室选育的A/Yunnan/1/2005 Vca(H3N2)毒株与WHO提供的疫苗生产用流行株A/Solomom Islands/3/2006(H1N1)进行传统遗传重配方法学的研究,以建立稳定而快速的遗传重配技术,并对重配株病毒reA/SI/2006(H1N1)Vca进行生物学表型特征的鉴定与疫苗安全性和免疫原性的研究,以证明供体株A/Yunnan/1/2005Vca(H3N2)能作为流感病毒Vero细胞减毒活疫苗的母本毒株。以两种病毒同时感染Vero细胞、MDCK细胞和鸡胚三种培养基质,加羊抗A/Yunnan/1/2005Vca(H3N2)病毒血清中和供体株,并在Vero细胞25℃条件下培养筛选,最终分别在Vero细胞33℃和MDCK细胞25℃成功重配获得能在Vero细胞25℃生长的毒株reA/SI/2006(H1N1)Vca。采用血凝抑制实验和单向免疫扩散试验对重配毒进行型别鉴定,结果证实重配株病毒抗原性与流行株A/Solomom slands/3/2006(H1N1)一致。进一步的基因测序结果表明,重配株reA/SI/2006(H1N1)Vca表面抗原基因HA和NA来自流行株A/Solomom Islands/3/2006(H1N1),6个内部基因来自供体株A/Yunnan/1/2005Vca(H3N2)。在经过优化的培养条件下,将重配毒株reA/SI/2006(H1N1)Vca在Vero细胞25℃连续传18代,病毒滴度在7.51gTCID50/mL以上,传代过程中病毒型别不发生改变。重配病毒reA/SI/2006(H1N1)Vca在25℃、33℃和39℃的感染性滴度分别为6.9、7.8和3.51gTCID50/mL,具有ca、ts表型。以106 TCID50的病毒量滴鼻接种BALB/c小鼠,免后连续7天测定小鼠鼻夹和肺组织的病毒载量,重配株病毒reA/SI/2006(H1N1)Vca组在前五天,上下呼吸道的病毒载量均比疫苗株低了至少31gTCID50/g,说明重配病毒具有减毒表型。以105TCID50、106TCID50和107TCID50三种剂量对BALB/c小鼠进行二次免疫,能有效刺激小鼠产生针对流行株病毒A/Solomom Islands/3/2006(H1N1)的血凝抑制抗体和中和抗体,并能促进粘膜sIgA的分泌。接种重配病毒reA/SI/2006(H1N1)Vca后,不仅完全保护小鼠免受同亚型流感病毒A/JN/2009(H1N1) 50MLD50病毒量的致死性攻击,而且能完全抑制攻击病毒在小鼠体内的复制。本研究表明,reA/SI/2006(H1N1)Vca是以遗传重配技术获得的既带有流行株的表面抗原基因,又能在Vero细胞25℃生长,并具有减毒表型特征的重配病毒,因此A/Yunnan/1/2005Vca(H3N2)可以用作流感病毒Vero细胞冷适应减毒活疫苗的母本株。
[Abstract]:Influenza virus is an acute respiratory infectious disease pathogen that seriously threatens human health. It causes high incidence and mortality in the world every year. Vaccination is the most effective means to prevent and control influenza epidemic. There are three main types of influenza vaccine approved at present: inactivated vaccine, live attenuated vaccine and recombinant HA vaccine. The only inactivated lysate vaccine or subunit vaccine is used. The vaccine can stimulate the body to produce humoral immunity and cause local mucosal immunity and cellular immune response. The immune effect is safe and effective. The preparation of the live attenuated influenza vaccine is mainly based on the gene of influenza virus. RNA, a segment of the segment, can be used to rematch the antivirus and epidemic strains by genetic matching. The redistribution plants obtain the surface antigen gene and the phenotypic characteristics of the antivirus strains from the epidemic strains. In this field, the most research is Russia and the United States, the United States adopts the cold adapted donor plant, A/Ann Arbor/6/60 (H2N2) and B/A, used by the United States. The first trivalent live attenuated influenza vaccine (Flumist), prepared by NN Arbor/1/66, was listed in the United States in 2003. At the same time, chicken embryo supply cycle is long, and when the pandemic attacks are difficult to meet mass production demand, especially when the avian influenza virus comes in, the chicken embryo is more affected. In view of this, WHO encourages the use of mammalian cells instead of chicken embryos as the substrate for influenza vaccine production..Vero cells are WHO approving producer vaccines. The cell line is not sensitive to influenza virus, and the yield of the virus is unstable. There is no seasonal influenza virus vaccine produced by Vero cells. It is very important to select cold adapted strains of influenza virus Vero cells for the research and development of the live attenuated influenza vaccine. At present, the cold adapted strains of Vero cells selected from all over the world are rare. Only one strain of A/Singapore/1/57ca (H2N2) was reported. In this laboratory, a Vero cell cold adapted influenza virus strain, A/Yunnan/1/2005Vca (H3N2), has been bred by gradient cooling culture, and has been passed on for 72 generations at 25 degrees centigrade. The titer of the virus is stable in the 7.8-8.21gTCID5o/mL. confirmed A/Yunnan/1/2005Vca (H3N2) with the cold adaptation phenotype (CA) and temperature. The degree sensitive phenotype (TS) and the attenuated phenotype (ATT), and the strain in ferrets and mouse models show good safety and immunogenicity, can effectively stimulate the body to produce cellular and humoral immunity, and protect the immune animals against the lethal attack of the same subtype influenza virus, suggesting that A/Yunnan/1/2005Vca (H3N2) may be prepared. An ideal candidate donor strain of a live attenuated influenza virus Vero cell vaccine strain. In this study, a laboratory selected A/Yunnan/1/2005 Vca (H3N2) strain and a popular strain A/Solomom Islands/3/2006 (H1N1) provided by WHO were used to study traditional genetic heavy prescription jurisprudence in order to establish a stable and rapid genetic redistribution technique, and The identification of biologic phenotypic characteristics and vaccine safety and immunogenicity of the reA/SI/2006 (H1N1) Vca of the recombinant strain show that the donor strain A/Yunnan/1/2005Vca (H3N2) can be used as the mother parent of the live attenuated vaccine of influenza virus Vero cells. Two viruses are simultaneously infected with Vero cells, MDCK cells and chicken embryos, plus three cultures. The Sheep anti A/Yunnan/1/2005Vca (H3N2) virus sera neutralized donor strain and cultured in Vero cells at 25 degrees centigrade. Finally, the strains of reA/SI/2006 (H1N1) Vca., which can grow at 25 degrees centigrade in Vero cells, were successfully rematched in Vero cells and MDCK cells at 25 degrees centigrade, respectively. The results showed that the antigenicity of the redistribution strain was the same as that of the epidemic strain A/Solomom slands/3/2006 (H1N1). Further gene sequencing results showed that the reA/SI/2006 (H1N1) Vca surface antigen gene HA and NA were derived from the A/Solomom Islands/3/2006 (H1N1) of the epidemic strain, and the 6 internal genes were derived from the donor plant A/Yunnan/1/2005Vca. Under the culture conditions, reA/SI/2006 (H1N1) Vca was transmitted for 18 generations in Vero cells at 25 degrees centigrade, the virus titer was above 7.51gTCID50/mL and the virus type did not change during the passage. The infective titers of the reA/SI/2006 (H1N1) Vca at 25, 33 and 39 were 6.9,7.8 and 3.51gTCID50/mL respectively, with Ca, TS phenotype. 106 The viral load of ID50 was inoculated in BALB/c mice, and the viral load of the nasal clips and lung tissues was measured for 7 days. The viral load in the upper and lower respiratory tract was lower than 31gTCID50/g in the first five days of the reA/SI/2006 (H1N1) Vca group, indicating that the redistribution virus had a detoxification phenotype. 105TCID50106TCID50 and 107TCID50 three were used. BALB/c mice were immunized with two times, which could effectively stimulate the mice to produce the hemagglutination and neutralizing antibodies against the epidemic strain A/Solomom Islands/3/2006 (H1N1), and promote the secretion of sIgA. After inoculation of reA/SI/2006 (H1N1) Vca, the mice were not only protected from the same subtype influenza virus A/JN/2009 (H1N1). This study shows that reA/SI/2006 (H1N1) Vca is a kind of 50MLD50 (H1N1) Vca, which is obtained by genetic redistribution technology with both the surface antigen gene of the epidemic strain, and can also grow at 25 degrees centigrade in the Vero cell, and has the characteristic of the attenuated phenotype. Therefore, A/Yunnan/1/2005Vc A (H3N2) can be used as the maternal strain of influenza virus Vero cell cold adapted live attenuated vaccine.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R392

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